1. Filed of the Invention
The present invention relates to a method of making a two-dimensional measurement of the concentration of a biological macromolecule solution, and an apparatus for use when carrying out this method. More particularly, the present invention relates to a method of observing the process of crystallization of a biological macromolecule, and an apparatus for two-dimensionally scanning the process of crystallization of the biological macromolecule when carrying out this method.
2. Description of the Related Art
Biological macromolecules such as proteins, polypeptides and nucleic acids exert important functions such as energy metabolism, structure construction and information transmission, and it is known that these functions of biological macromolecules result from the structures thereof, and therefore, the need for an investigation of the three-dimensional structure of a biological macromolecule has increased. As a method of analyzing this three-dimensional structure of the biological macromolecule, in general the NMR (nuclear magnetic resonance) method, the X-ray diffraction method, and the like are adopted, although the X-ray diffraction method is now attracting attention because it enables the three-dimensional structure to be directly analyzed.
To accomplish the X-ray diffractometry, it is important that a biological macromolecule crystal having a structure reduced disorder and a high quality be obtained, but it is difficult to obtain a high-quality crystal of a biological macromolecule because the crystallization conditions differ according to respective biological macromolecules, and the optimum conditions must be determined by trial and error. Furthermore, since knowledge about the growth process conditions of crystal nuclei of biological macromolecule crystals is limited, it is difficult to obtain a biological macromolecule crystal having a high quality.
Recently, trials have been initiated to examine in detail the growth process of a crystal of a biological macromolecule, especially a protein (see, for example, Azuma et al., J. Crystal Growth, 98, 371-376, 1989). In this thesis, the process of crystallization of a protein is traced by measuring changes of the refractive index in the vicinity of the crystal, by utilizing a Mach-Zehender interference optical system. According to this method, it becomes possible to perform a two-dimensional measurement of the concentration gradient in the diffusion foundary layer around a growing crystal as a function of concentration of the lysozyme concentration, but the method of measuring the protein concentration by the differential interference optical system has several problems. First, since the protein concentration is measured, based on the refractive index, there is a risk of mistaking a change of the solute concentration in a crystal mother liquor for a change of the protein concentration, and second, since for a conversion of the refractive index to the protein concentration it is necessary to measure the space between interference fringes in the vicinity of the crystal and the mother liquor having a concentration gradient in the diffusion boundary layer around a growing crystal interface, a complicated operation such as image processing is necessary by transforming the refractive index gradient using the Lorentz-Lorentz formula.
A two-dimensional measuring apparatus utilizing ultraviolet rays recently has been developed as an analysis and measurement apparatus. As the two-dimensional measurement apparatus utilizing ultraviolet rays, there can be mentioned, for example, an ultraviolet microscope. An apparatus is known which is constructed by combining this ultraviolet microscope with a microscope photometer (MPM) system, so that the intercellular calcium concentration and the DNA quantity can be measured. For example, there can be mentioned the MPM System supplied by Carl Travis Co. This apparatus, however, is not designed for the analysis of a biological macromolecule crystal, and it has to comprise a heavy, large, and delicate specific optical system. Thus, although a point light measurement by a microscope system can be performed, it is not possible to promptly perform a light measurement over a wide range.
Accordingly, the development of a method of two-dimensionally tracing the crystallization process of a biological macromolecule, easily and promptly, and of measuring the concentration of a biological macromolecule concentration, and a simple and small apparatus for carrying out this method, is desired.